The extracellular matrix (ECM) is a dynamic assemblage of interacting molecules that regulate cell functions and interactions in response to stimulation. One class of extracellular matrix macromolecules, the glycosaminoglycans, are molecules known to be involved in a wide array of both normal and abnormal biological processes, including cell migration, differentiation, proliferation, immune response and cytoskeletal organization.
The glycosaminoglycan hyaluronan (HA) is a repeating disaccharide of [GlcNAcβ1-4GlcUAβ1-3]n that exists in vivo as a high molecular weight linear polysaccharide. HA is found in mammals predominantly in connective tissues, skin, cartilage, and in synovial fluid, and is also the main constituent of the vitreous of the eye. In connective tissue, the water of hydration associated with HA creates spaces between tissues, thus creating an environment conducive to cell movement and proliferation. HA plays a key role in biological phenomena associated with cell motility including rapid development, regeneration, repair, embryogenesis, embryological development, wound healing, angiogenesis, and tumorigenesis (Toole, Cell Biol. Extracell. Matrix, Hay (ed), Plenum Press, New York, 1384-1386 (1991); Bertrand et al. Int. J. Cancer 52:1-6 (1992); Knudson et al, FASEB J.7:1233-1241 (1993)). HA levels have been shown to correlate with tumor aggressiveness (Ozello et al., Cancer Res. 20:600-604 (1960);
Takeuchi et al., Cancer Res.36:2133-2139 (1976); Kimata et al., Cancer Res.43:1347-1354 (1983)), and can be indicative of the invasive properties of tumor cells. M. M. Knupfer et al., Anticancer Res 18:353-6 (1998).
HA is also involved in immune response, and thus may mediate this response in both normal and abnormal biological reactions. Increased binding of HA to one of its receptors, CD44, has been shown to mediate the primary adhesion (“rolling”) of lymphocytes to vascular endothelial cells under conditions of physiologic shear stress, and this interaction mediates activated T cell extravasation into an inflamed site in vivo in mice. H. C. DeGrendele, et al., J. Exp. Med.183:1119-1130 (1996); H. D. DeGrendele,et al., Science 278:672-675 (1997). H. C. DeGrendele et al., J. Immunol.159:2549-2553 (1997). Alterations in levels of HA and other glycosaminoglycans have also been associated with unwanted immune responses, and may be involved in diseases and disorders such as rheumatoid arthritis, atopic dermatitis, psoriasis, multiple sclerosis, transplantation rejection. For example, HA and other glycosaminoglycans display are altered in autoimmune disorders such as arthritis, and decreased levels of both hyaluronic acid and chondroitin 6-sulfate have been found in the diseased synovial fluid of both adults with rheumatoid arthritis (A. Bensouyad et al., Ann Rheum Dis 49:301-7 (1990)) and children with juvenile rheumatoid arthritis (P. F. Spelling et al. Clin Exp Rheumatol 9:195-9 (1991)).
Dendritic cells (DC) play essential roles in the induction of cellular immune responses to a variety of relevant antigens. DC are known to play critical roles in the induction of cellular immune responses against a wide variety of antigens of relevance, including chemical haptens, foreign proteins, infectious microbes, and tumor-associated antigens (Steinman, “The dendritic cell system and its role in immunogenicity.” Ann. Rev. Immunol.9:271 (1991); Stingl et al., “The Epidermis: An Immunologic Microenviromnent In Dermatology in General Medicine.”T. B. Fitzpatrick, ed. McGraw Hill and Co., New York, p. 172 (1993)). Interaction between HA, expressed on endothelial cells, and CD44, expressed on activated dendritic cells as well as T cells, and granulocytes, is believed to mediate homing of such leukocytes to their target sites.
Glycosaminoglycans, and particularly HA, are also known to mediate other cellular interactions that involve binding and entry into a cell. For example, HA is involved in infection of mammalian cells by the Human Immunodeficiency Virus (HIV), since HIV is known to bind to HA upon infection. Both HA and monoclonal antibodies to its receptor CD44 were found to inhibit HIV infection of monocytes by monocytotropic HIV. M. C. Levesque and B. F. Haynes, J. Immunol 156:1557-65 (1996). HA is also involved in mammalian zygote formation by mediating binding of the oocyte and the sperm. Data suggests that HA in the cumulus matrix may act to prime the fertilizing sperm for induction of the acrosome reaction by constituents of the cumulus and/or zona pellucida. HA is thought to mediate this interaction by binding to the PH-20 protein to increase basal levels of intracellular calcium and thereby potentiate the acrosome reaction. K. Sabeur et al., Zygote 6:103-11 (1998). HA mediates sperm motility by enhancing phosphorylation of proteins including HA binding protein. S. Ranganathan et al., Cell Mol Biol Res 41:467-76 (1995).
The role of glycosaminoglycans, and particularly hyaluronic acid, in such varying physiological processes make them attractive targets for therapeutic agents. Unfortunately, glycosaminoglycans have been found to be nearly non-antigenic, and very few antibodies that recognize glycosaminoglycans have been isolated. Due to this lack of antigenicity, it has been technically difficult to develop inhibitors or probes of glycosaminoglycans. Thus, there is a need in the art for inhibitors of glycosarninoglycan-mediated processes, and in particular for inhibitors of hyaluronic acid-mediated processes. There is also a need for a method of identifying effective glycosaminoglycan inhibitors in a systematic, reproducible manner.